A method for detecting the antibodies to replication-competent retrovirus (RCR) was developed. Specific fragments of murine leukemia virus (MLV) Gag or Env protein were cloned and expressed in Escherichia coli, and used subsequently to develop the ELISA system. It was found that CA of Gag and SU of Env, but not the transmembrane portion of Env, could be used in ELISA. ELISA conditions such as coating buffer and blocking solution were optimized using sera obtained from mice immunized with amphotropic MLV particles. In an optimized ELISA system, serum samples from normal healthy individuals provided very low absorbance values. ELISA was performed using serum samples from patients who had received skin fibroblasts engineered with MLV-based retroviral vector. Experimental samples presented absorbance values comparable to those found with control serum samples from normal, healthy individuals, showing no evidence of RCR infection.